Hepatitis C virus (HCV) infections affect approximately 3 percent of the worldwide population and often lead to cirrhosis and hepatocellular carcinoma. The current therapy of pegylated-interferon and ribavirin induces serious side effects and provides viral eradication in less than 50% of patients.
microRNAs (miRNAs) are small regulatory RNAs that play important roles in development and disease1-3 and, thus, represent a potential new class of targets for therapeutic intervention4. Despite recent progress in silencing of miRNAs in rodents5,6, the development of effective and safe approaches for sequence-specific antagonism of microRNAs in vivo remains a significant scientific and therapeutic challenge. Moreover, there are no reports of miRNA antagonism in primates. Here we show that simple systemic delivery of an unconjugated, saline-formulated Locked Nucleic Acid-modified oligonucleotide (LNA-antimiR) effectively antagonizes the liver-expressed microRNA-122 in non-human primates. Acute administration by intravenous injections of 3 or 10 mg/kg LNA-antimiR to African green monkeys resulted in uptake of the LNA-antimiR in the cytoplasm of primate hepatocytes and formation of stable heteroduplexes between the LNA-antimiR and miR-122. This was accompanied by depletion of mature miR-122 and dose-dependent lowering of plasma cholesterol. Efficient silencing of miR-122 was achieved in primates by three doses of 10 mg/kg LNA-antimiR leading to long-lasting and reversible reduction of total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals. Our findings demonstrate the utility of systemically administered LNA-antimiRs in exploring microRNA function in rodents and primates and support the potential of such compounds as a novel class of therapeutics for disease-associated microRNAs. MiR-122 is a liver specific microRNA, well conserved within vertebrates. MiR-122 is involved in cholesterol metabolism (Esau et al. 2005) and it has recently been shown that miR-122 is important for hepatitis C(HCV) replication in cultured cells (Jopling et al. 2005).
The sequence of miR-122 is well conserved between different mamalian species (mirbase, Sanger Center, UK).
>hsa-miR-122 MIMAT0000421UGGAGUGUGACAAUGGUGUUUG(SEQ ID NO: 1) >mmu-miR-122 MIMAT0000246UGGAGUGUGACAAUGGUGUUUG(SEQ ID NO: 1) >rno-miR-122 MIMAT0000827UGGAGUGUGACAAUGGUGUUUG(SEQ ID NO: 1) >dre-miR-122 MIMAT0001818UGGAGUGUGACAAUGGUGUUUG(SEQ ID NO: 1) >xtr-miR-122 MIMAT0003585UGGAGUGUGACAAUGGUGUUUGU(SEQ ID NO: 2) >gga-miR-122 MIMAT0001190UGGAGUGUGACAAUGGUGUUUGU(SEQ ID NO: 2) >bta-miR-122 MIMAT0003849UGGAGUGUGACAAUGGUGUUUG(SEQ ID NO: 1)
Joplin et al. also show that blocking miR-122 by an oligonucleotide inhibits HCV genomic replication in vitro. miR-122 interacts with a target sequences in the 5′UTR and 3′UTR of the virus, mutations in these sites reduce virus replication. Both the 5′UTR and 3′UTR miR-122 target site is conserved in the HCV genotypes 1a, 1b, 2, 3, 4, 5, and 6. This suggests that all HCV genotype replication can be reduced by blocking miR-122. It has also recently been shown that genotype 2 replication is blocked by targeting miR-122 with a complementary oligonucleotide (Randall et al. PNAS 2007). About 70% of all infected establish chronic infection. Current antiviral therapy against HCV consists of interferon in combination with ribavirin, which is effective in some patients, however a large group do not respond or do not tolerate the treatment. Therefore, a need for novel treatment modalities for HCV exists. The present invention provides a novel treatment for HCV patients that are not responding to treatment by interferon, wherein an effective dose of an inhibitor of miR-122 is provided to the patient. The treatment provided leads to a reduction of an endogenous host factor miR-122 that is needed for the full replication of HCV, and furthermore amend the expression of IRG transcripts.
A large proportion of HCV infected patients do not respond to treatment with interferon, why there is a need for novel treatment modalities that can either replace the interferon treatment, or alternatively make the patient respond to such treatment. An inverse relationship has been observed between the pretreatment hepatic levels for some IRG, e.g., interferon stimulated gene transcripts and the virologic response to therapy (Chen et al. 2005, Gastroenterology, 128; 1437-1444). In particular, in livers of HCV infected Chimpanzees that lack an antiviral response to interferon, a lack of an Interferon-Gamma-Inducible Protein-10 (IP-10 or CXCL10) transcriptional response to IFN in the liver can be observed (Lanford et al. 2007, Hepatology, vol 46, 999-1008). Moreover, Lanford showed that the baseline levels of IP-10 transcripts in the HCV-infected chimpanzees were significantly higher than the baseline levels in the uninfected animals. In more general, Lanford show lack of induction of several IRGs, referred to therein as interferon stimulated genes, in HCV infected livers. Ribavirin has been shown by Feld et al.26 2010 to enhance the response to PEG-interferon, due to an enhanced induction of IRG's. However, the size of the enhanced response that was seen when Ribavirin was combined with Interferon, correlated with the size of the initial response to Interferon when administered alone (in terms of decrease in HCV RNA), so that a poor response was enhanced less effective than a good response to Interferon was. Since a poor initial response to Interferon treatment in a patient, will result in a relatively poor enhancement of the response if Ribavirin is added to the treatment. Therefore, Ribavirin is not the solution to the problem of providing a compound or a class of compounds that will make non-responders to Interferon change to become responsive, since the Interferon non-responders (whether they exhibit a low response or no response at all) will have lesser benefit of Ribavirin as compared to the benefit experienced by normal responders.
While interferon alpha is a major treatment of choice in HCV infected patients, it is also important for the treatment of recurrent hepatitis C in liver transplant recipients. However, one of the potential serious adverse effects of such treatment with interferon is acute and chronic rejection and subsequent graft loss (Walter et al. 2007, Americal Journal of Transplantation, 7, 177-184). HCV recurrence after transplantation is almost universal, and HCV infection impairs patient and allograft survival. The course of HCV graft disease is accelerated in transplant recipients, compared with immune-competent patients (review: Samuel, Liver Transplantation, Vol 10, No 7 (July), 2004: pp 868-87). High expression of the interferon regulated IP-10 (CXCL10) has been shown to be prognostic for kidney transplant rejection (Matz et al. Kidney International (2006) 69, 1683-1690). Krukemeyer et al. (2004, Transplantation, vol 78, 65-70) has demonstrated an elevated expression of IP-10 in rejected liver grafts. Therefore, there is a need for treatments that will modulate IRG transcript expression, for example IP-10, towards normalization, in cells, such as in transplanted organs, i.e. transplanted livers, i.e. in transplanted livers of HCV patients.
Further, the findings of the present invention shows that pharmaceutical compositions comprising an anti microRNA-122 oligonucleotide of the invention, may be made and wherein the composition is made for administration to a cell, such as a primate cell, and wherein the administration causes the expression levels of IRGs, such as IP-10 (CXCL10), to be altered towards normal levels, to prevent non-responsiveness to interferon in HCV infected individuals, or to prevent organ transplant rejection.